This invention relates generally to the field of bacterial antigens and their use, for example, as immunogenic agents in humans and animals to stimulate an immune response. More specifically, it relates to the vaccination of mammalian species with heteropolymeric protein complexes as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species, including all types of enterobacteriacea.
In a particular aspect, the present invention relates to the prevention and treatment of urinary tract infections such as cystitis and/or pyelonephritis that are caused by pilus-carrying bacteria (uropathogenic bacteria). In this regard, certain types of Escherichia coli are of particular interest since they are the predominant etiologic agent of urinary tract infections (UTIs).
UTIs provide one example of a disease process that is mediated (or assisted) by the attachment to bacteria to cells. E. coli is the most common pathogen of the urinary tract, accounting for greater than 85% of cases of asymptomatic bacteriuria, acute cystitis and acute pyelonephritis, as well as greater than 60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis infections. Furthermore, approximately 25%-30% of women experience a recurrent E. coli urinary tract infection within the first 12 months following an initial infection: after a second or third infection the rate of recurrence increases to 60%-75%. Given the high incidence, continued persistence, and significant expense associated with E. coli urinary tract infections, there is a need for a prophylactic vaccine to reduce susceptibility to this disease.
While many factors contribute to the acquisition and progression of E. coli urinary tract infections, it is widely accepted that colonization of the urinary epithelium is a prerequisite to infection. In a typical course of E. coli urinary tract infection, bacteria originate from the bowel, ascend into the bladder, and adhere to the bladder mucosa where they multiply and establish an infection (cystitis) before ascending into the ureters and kidneys. Disruption or prevention of pilus-mediated attachment of E. coli to urinary epithelia may prevent or retard the development of urinary tract infections. In this regard, a number of studies have pointed to a role for pili in mediating attachment to host uroepithelial cells.
To initiate infection bacterial pathogens must first be able to colonize an appropriate target tissue of the host. For many pathogens this tissue is located at a mucosal surface. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Attachment is mediated via proteins on the bacterium that bind specifically to cellular receptors. These proteins, or adhesins, are expressed either directly on the surface of the bacterium, or more typically, as components of elongated rod-like protein structures called pili, fimbriae or fibrillae.
Type 1 pili are thought to be important in initiating colonization of the bladder and inducing cystitis, whereas P pili are thought to play a role in ascending infections and the ensuing pyelonephritis.
Such pili are heteropolymeric structures that are composed of several different structural proteins required for pilus assembly. Two types of pili are of particular interest: P pili and type 1 pili. P pili-carrying bacteria recognize and bind to the galxcex1(1-4)gal moiety present in the globoseries of glycolipids on kidney cells in mammals. Type 1 pili-carrying bacteria recognize and bind to D-mannose in glycolipids and glycoproteins of bladder epithelial cells.
PapG, the adhesin protein in P pili bacteria that mediates the specific interaction of the pilus with receptors on the surface of host cells, is found at the distal end of the tip fibrillum. Its periplasmic chaperone protein is PapD which is highly conserved across strains of E. coli. (Hultgren et al., Proc. Natl. Acad. Sci. USA 86:4357 (1989); EMBO Journal 15:3792-3805 (1996).
With regard to type 1 pili, tip adhesins and other ancillary subunits also have been identified. FimH is the D-mannose-binding adhesin that promotes attachment of type 1-piliated bacteria to host cells via mannose-containing glycoproteins on eukaryotic cell surfaces. FimC is its periplasmic chaperone protein. FimH is also highly conserved not only among uropathogenic strains of E. coli, but also among a wide range of gram-negative bacteria. For example, all Enterobacteriacea produce FimH. Thus, vaccines incorporating the FimH antigen should exhibit a broad spectrum of protection.
Chaperone proteins are a class of proteins in gram-negative bacteria that are involved in the assembly of pili by mediating such assembly, but are not incorporated into the structure. PapD is the periplasmic chaperone protein mediating the assembly of pili for P piliated bacteria and FimC is the periplasmic chaperone protein that mediates assembly of type 1 pili in bacteria.
Antibodies directed against purified whole type 1 or P pili protect against cystitis and pyelonephritis, respectively, in both murine and primate models for these diseases. Abraham et al., Infect Immun. 48:625 (1985), Roberts et al., Proc. Natl. Acad. Sci. (USA) 91:11889 (1994), O""Hantey et al., J. Clin. Invest. 75: 347 (1985). However, such protection is limited to either homologous E. coli strains from which the pili used as immunogens were derived, or to a small subset of serologically cross-reactive heterologous strains. Therefore, vaccines composed predominantly of the major structural proteins of pili (i.e., PapA or FimA) appear to be of limited value because antibodies developed against these highly variable proteins are specific for the strains used for immunization.
In one aspect the present invention relates to a vaccine for treating or preventing bacterial infections which utilizes as an immunogen a complex of a bacterial periplasmic chaperone protein with a bacterial adhesin protein. Preferably, the adhesin protein is a pilus adhesin protein. In one embodiment, the periplasmic chaperone protein and pilus adhesin protein are from E. coli; for example, a member selected from the complexes PapD/PapG and FimC/FimH.
In a particular aspect, the invention relates to vaccines formulated from type 1 pilus-associated adhesins (or from mannose-binding fragments thereof) or from complexes of chaperone proteins (including PapD-like chaperones) and pilus-associated adhesins for the treatment and/or prophylaxis of diseases caused by pathogenic species of gram-negative bacteria, such as Escherichia coli (E. coli). For example, it relates to treatment and/or prophylaxis of urinary tract infections caused by E. coli with vaccines formulated from at least one of (1) a fragment of the pilus-associated adhesin FimH that retains mannose binding capability (alone or complexed with its chaperone FimC), (2) the pilus-associated adhesin PapG protein complexed with its periplasmic chaperone protein PapD or (3) the full-length pilus-associated adhesin FimH (alone or in a complex with its chaperone protein FimC). This invention also relates generally to the use of heteropolymeric protein complexes to raise antibodies in non-human mammalian species useful, for example, as diagnostic reagents and vaccines.
In yet another aspect, the present invention relates to the production of essentially full-length bacterial adhesin proteins in a recombinant host (in E. coli, another bacterial species, a bacterial species with one or more disabled proteases, or a non-bacterial production vector or host cell) or by synthesis. Such recombinant or synthetic methods permit the production of the full-length adhesin protein in the presence or absence of its chaperone protein and when chaperones are absent preferably in the absence of proteases that will shorten its length or start to break it down. Even more preferable is the production of essentially full-length FimH or a mannose binding analog or variant for use as a vaccine. If such recombinant production is performed in a host which is capable of producing an usher protein, the recombinant production is under conditions which eliminate usher production.
Using novel methods disclosed herein, it is possible to recombinantly introduce into a bacterial cell the Fimh and FimC genes using a single vector, commonly a plasmid but in no way limited thereto.
In a yet further aspect, the present invention relates to the production of mannose-binding fragments of bacterial adhesin proteins in a recombinant host (in E. coli, another bacterial species, or a non-bacterial production vector or host cell) or by synthesis. Preferably, when such protein fragments are expressed in a bacterial host that produces an usher protein they are expressed under such conditions that their usher protein is not expressed. Such recombinant or synthetic methods permit the production of mannose-binding adhesin protein fragments in the absence of the their chaperone protein or as a complex with their chaperone protein that can later be separated from the chaperone protein. Preferably, such fragments are produced under conditions that avoid shortening of their length by cleavage or break down by proteases.
In another aspect, the proteins are produced with a histidine label (or other suitable label) such that the full-length proteins or fragments can be isolated due to their label.
In a further aspect, the present invention relates to the production of a periplasmic chaperone protein in a complex with an essentially full-length bacterial adhesin protein or appropriate fragment thereof in a recombinant host (in E. coli, another bacterial species, a bacterial species with one or more disabled proteases, or a non-bacterial production vector or host cell) or by synthesis or by recovering from a natural source. Even more preferable is the production of the periplasmic chaperone protein FimC complexed with essentially full-length FimH or a mannose binding analog or variant for use as a vaccine.
In another aspect the present invention relates to a method of prophylaxis and/or treatment of diseases that are mediated by pili-bearing bacteria that have adhesin proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by type 1 pili adhesin proteins. In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of UTIs in humans, particularly in women or children.
In still another aspect the present invention relates to a method of using one or more antibodies (monoclonal, polyclonal or sera) to either a periplasmic chaperone protein or fragment thereof complexed with an adhesin protein or an adhesin protein (alone) for the prophylaxis and/or treatment of diseases that are mediated by pili-bearing bacteria that have adhesin proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by type 1 pili adhesin proteins. In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of UTIs in humans, particularly in women or children, by utilizing antibodies to either the periplasmic chaperone protein FimC complexed with the adhesin protein FimH (anti-FimC-H) or the adhesin protein FimH alone (anti-FimH).